1. Field of the Invention
The present invention relates to a technique for detecting a target nucleic acid, more particularly to a method for detecting a target nucleic acid sequence present in a LAMP amplified product and a LAMP amplified product for use in detection of a target nucleic acid.
2. Description of the Related Art
To select correct therapeutic agents, it is important to identify bacteria causing a given infection. Currently, culture method and nucleic acid amplification method are used for microorganism tests.
Recently, improvement in culture method has been made in an effort to improve detection sensitivity and to reduce days of culture. Immunochromatography utilizing antigen-antibody reactions rapidly becomes popular as a simple identification method (see, for example, Japanese Patent Publication No. 2001-103981 (paragraph 0011-0012) and Japanese Patent Publication No. 2002-125695 (paragraph 0002)). However, since this method still takes several weeks for bacterial growth, it hardly meets the needs sought in clinical settings. Consequently, patients are often given a wrong treatment until identification results are obtained.
In contrast, the nucleic acid amplification method uses specific primers to identify bacterial species or resistant bacteria by investigating presence or absence of amplification. This method, including a sample preparation step, can provide test results within approximately 6-7 hours, making it a very useful rapid test. Furthermore, under the recent genomic analysis competition, entire genetic information of many organisms are being analyzed, and as a result, this method is expected to become available for identification of a wide range of bacterial species.
A method well-known as a nucleic acid amplification technique is PCR method (Polymerase Chain Reaction method, Roche). PCR method is widely used as a tool for genetic analyses such as gene cloning and structural determination. However, PCR method has a disadvantage of requiring a complex temperature control device like Thermal Cycler and reaction time over two hours. In PCR method, If synthesis of wrong complementary chains occurs by any chance, the resultant products work as templates for amplification, thereby leading to a wrong identification. In fact, it is difficult to regulate specific amplification based on a difference of only one base in a primer terminal.
In addition, due to the fact that double-stranded products are generated in general when target gene products amplified by PCR method are detected by a DNA chip, complementary strands work as competitors for probes upon a hybridization reaction with the probes, reducing hybridization efficiency and detection sensitivity. To address this problem, such methods as digesting or separating complementary strands have been employed to turn a target into a single-stranded sequence. However, this method has still several problems including a need to use enzymes, expensiveness due to the use of magnetic beads, and handling complexity.
LAMP method (Loop-mediated isothermal amplification method) has been developed as a gene amplification method to fix this problem. LAMP method completes gene amplification within one hour under an isothermal condition. Also, 100-1,000 times amount of final products are obtained compared to that of PCR products. LAMP method also has an advantage of higher specificity as compared to PCR method because six primer regions are set in LAMP method (see, for example, U.S. Pat. No. 3,313,358). LAMP method is expected to be a promising technique to rapidly detect bacteria, viruses and gene mutations (see, for example, Japanese Patent Publication No. 2003-159100) with high sensitivity.
Among tests using LAMP method, a method is now commercialized which detects presence or absence of gene amplification by measuring white turbidity of magnesium pyrophosphate, a by-product which is produced in the course of amplification (see, for example, Japanese Patent Publication No. 2003-174900). In this method where white turbidity of a by-product (magnesium pyrophosphate) is measured, there is no way of confirming whether amplification of an unintended product occurs or not. LAMP method has also a problem that it cannot detect multiple target genes simultaneously.
In addition, methods using intercalators or optical properties have been known as procedures for detecting LAMP amplified products (see, for example, Japanese Patent Publication No. 2002-186481). Other known methods are those measuring degree of fluorescence polarization of reaction solution by fluorescently labeling a probe which hybridizes to a single-stranded loop portion present in a LAMP product (see, for example, Japanese Patent Publication 2002-272475) as well as those immobilizing an insoluble carrier to a 5′ terminal side of a primer which hybridizes to a single-stranded loop portion and observing an aggregation reaction associated with amplification reaction (see, for example, Japanese Patent Publication 2002-345499).